1. Field
The present invention relates generally to the field of peptides reactive with antibodies directed against HPV. Some have termed this type of peptide as antigenic or immunoreactive. More particularly, the invention relates to peptides derived from the early coding region of the E2, E6, and E7 oncoproteins of human papillomavirus [HPV] and a method for their use for the diagnosis of HPV associated epithelial cell abnormalities via an immunoassay.
2. State of the Art
The human papillomaviruses (HPV), named because certain types induce warts or papillomas, cause virtually all cervical cancers (Nobbenhuis et al., xe2x80x9cRelation of human papillomavirus status to cervical lesions and consequences for cervical-cancer screening: a prospective studyxe2x80x9d, The Lancet, 354:20-25, 1999; Cuzick et al., xe2x80x9cA systematic review of the role of human papilloma virus (HPV) testing within a cervical screening programme: summary and conclusionsxe2x80x9d, British Journal of Cancer, 83:561-565, 2000). These encompass not only squamous cell carcinomas (Nobbenhuis et al., 1999) but also adenocarcinomas (Pirog et al., xe2x80x9cPrevalence of human papillomavirus DNA in different histological subtypes of cervical adenocarcinoma,xe2x80x9d American Journal of Pathology, 157:1055-1062, 2000). These viruses are also strongly associated with vulvar and vaginal carcinomas (Frisch et al., xe2x80x9cHuman papillomavirus-associated carcinomas in Hawaii and the mainland USxe2x80x9d, Cancer 88:1464-1469, 2000; Sugase et al., xe2x80x9cDistinct manisfestations of human papillomaviruses in the vaginaxe2x80x9d, International Journal of Cancer, 72:412-415, 1997), as well as cancers of the anus (Frisch et al., 2000) and penis (Gregoire et al., xe2x80x9cPreferential association of human papillomavirus with high-grade histologic variants of penile-invasive squamous cell carcinomaxe2x80x9d, Journal of the National Cancer Institute, 87:1705-1709,1995). Moreover, HPV may be responsible for certain carcinomas in the head and neck region (Mellin et al., xe2x80x9cHuman papillomavirus (HPV) DNA in tonsillar cancer: clinical correlates, risk of relapse, and survivalxe2x80x9d, International Journal of Cancer, 89:300-304, 2000; Zumbach et al., xe2x80x9cAntibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in patients with head-and-neck squamous-cell carcinomaxe2x80x9d, International Journal of Cancer, 85:815-818, 2000), seem associated with the more deadly melanomas (Dreau et al., xe2x80x9cHuman papilloma virus in melanoma biopsy specimens and its relations to melanoma progressionxe2x80x9d, Annals of Surgery, 231:664-671, 2000), and could play a role in lung carcinomas (Soini et al., xe2x80x9cPresence of human papillomavirus DNA and abnormal p53 protein accumulation in lung carcinomaxe2x80x9d, Thorax 51:887-893, 1996) and perhaps other cancers. HPV exist as different genetic types, designated by numbers, concerning which only a subset is oncogenic or cancer causing. Over 100 HPV genotypes have been identified. Cancers stem overwhelmingly from HPV 16 and 18 but also from types 31, 33, 35, 45, 51, 52, 56 and 58. The virus infects cervical and other cells that can support virus propagation, where it causes abnormal cellular changes that can lead to life threatening malignancies. Cervical cancer is the second most common cancer among women worldwide. Each year, about 450,000 women worldwide are diagnosed with cervical cancer, and nearly 300,000 women die of this disease. Since the advent of organized cervical cancer screening via cytology 50 years ago, the mortality rate of cervical cancer has dramatically decreased in developed countries. In fact, cervical cancer can be considered preventable. The key to prevention is the timely identification and management of precancerous lesions through accessible and affordable screening programs. At present, 11.8% of global cancer incidence in females is due to HPV infections of the cervix. There is consensus that oncogenic HPV detection would be an effective way to identify cancer victims or those at high risk for the disease. Notably, HPV detection would facilitate early detection, when cancer would exist at a more readily curable stage.
HPV infection requires cells able to replicate their DNA, specifically those in the basal epidermal layer. Entry occurs through microlesions that expose basal proliferating cells to the surface. The virus attaches to a cell surface receptor and gains entry into the cytosol. The infecting virus particle contains a closed-circular double-stranded DNA genome of 7000 to 8000 base pairs composed of eight early transcribed open reading frames, E1 to E8, which are unequally represented among HPV genotypes, two late open reading frames, and a noncoding long control region.
Much has been discovered about how HPV DNA integrates into host chromosomes and how the E1 and E2 oncoproteins are involved with this process. Its relevance to immunological diagnostics is that antibodies against E1 and E2 gene products comprise evidence that HPV infection has occurred.
The manner by which infection by HPV leads to cancer centers about the E6 and E7 gene products. In host cells, these form complexes with the cellular p53 and retinoblastoma tumor suppressing proteins regulating cell division. By functionally neutralizing or inactivating these proteins, cells enter into the S phase of the cell cycle. The E7 oncoprotein further destabilizes cell control through its interaction with the cyclin-dependent kinase inhibitor protein, p21. These interactions set the stage for controlling host cell proliferation and differentiation (i.e., transformation), a first step in the conversion of normal cells to preneoplastic ones and ultimately to the full expression of malignancy.
The E6 and E7 oncoproteins are constitutively expressed in tumor cells, and silencing these genes yields reversion of the malignant phenotype. Thus, the E6 and E7 gene products seem tumor-specific antigens, and possible targets or probes for antibodies in immunological cancer tests as well as antigens in vaccines for controlling HPV induced tumors.
Indeed, the E6 and E7 oncoproteins appear natural targets for antibody production due to their consistent expression in cervical cancer cells. The response against the E7 one in earlier studies had only been moderately disease specific, but E7 IgG and IgA have now been verified as strongly disease associated. Antibodies against the E6 and E7 oncoproteins are at high levels in sera from cervical cancer patients compared against non-cancer controls. Moreover, such antibodies seem detectable by immunological means even when present in lesser amounts. Sensitivity for identifying HPV infections and possible cancers increases with a combination of serological tests of multiple virus proteins. Hence, using both oncoproteins yields positive immunological results with samples from cervical cancer patients.
The main method for public health screening for cervical cancer has been the Papanicolaou smear. For a variety of reasons, the Papanicolaou smear is less than an ideal screening test. Drawbacks include difficulty of obtaining samples, high rate of false negatives (up to 20%), and requirements for specialized labs staffed by highly trained personnel. Nucleic acid methods have been developed, but are not ideal primarily due to their high cost and like requirement for highly trained personnel. Another assay is the so-called xe2x80x9cDNA Hybrid Capturexe2x80x9d. This method suffers from high cost and sampling difficulties. What is needed is a low cost, simple, sensitive and specific assay that can be performed on readily obtainable bodily samples.
An object of the invention is to develop antibody active peptides derived from the HPV E2 protein and the HPV 16 and 18 E6 and E7 oncoproteins. It is a further object to provide these peptides in a chemically pure form. It is a still further object to provide a simple, rapid, less expensive and more sensitive test for diagnosing not only HPV infections, but also most, if not all, HPV associated neoplasms. A further object is to provide antigens for use in HPV inoculums that will induce antibody production and killer T cell activity.
The above stated objects and other objects of the invention are accomplished by novel peptides, the sequences of which were derived by the inventor from careful analysis of the early coding regions of the E2, E6, and E7 oncoproteins of HPV 16 and 18. The peptides lend themselves to a highly sensitive and specific diagnostic immunoassay. Antibodies to the E2 oncoprotein are found in those infected with HPV. Antibodies to the E6 and E7 oncoproteins are found in those with HPV associated neoplasms. The peptides of the invention, ranging in size from about 19 amino acid residues to about 30 amino acids can readily be synthesized by chemical means and obtained at purities that can exceed 99%. Although the peptides could be obtained by other means, in their pure form there may be a much reduced likelihood for undesirable cross reactivity with random antibodies. Hence, the pure peptides of the invention lend themselves to diagnostic immunoassays of high specificity. The diagnostic immunoassay method comprises taking a sample of body fluid or tissue likely to contain antibodies, if present, reacting it with one or more of the peptides of the invention, then assaying for the presence of an antibody-peptide reaction.
Immunoassays employing peptides derived from the E2 region serve as reliable indicators that HPV infection has or has not occurred. Immunoassays employing the peptides derived for the E6 and E7 oneoproteins serve as reliable indicators that HPV associated malignancy or premalignant cell transformation has taken place. One of the most useful aspects of the invention is in diagnosing cervical carcinoma, both squamous cell and adenocarcinoma as well as any epithelial cell abnormality associated with oncogenic HPV infection including koilocytosis; hyperkerotosis; precancerous conditions encompasssing intraepithelial neoplasias or intraepithelial lesion; high-grade dysplasias; and invasive or malignant cancers. Besides cervical cancer, detection of antibodies to peptides derived from the HPV E6 and E7 oncoproteins is useful for detecting head and neck cancers, small cell lung cancers, penal and anal squamous cell carcinomas, and melanoma.